Chronic pain is a widespread problem, estimated to affect 1 in 3 Americans. An important component of neuropathic pain is spontaneous or ongoing pain, i.e. pain without stimulus that may result from abnormal spontaneous activity in sensory nerves. This includes ongoing burning pain and intermittent paroxysms of sharp, shooting pain. Spontaneous activity has been reported in cultured neurons and acutely dissected sensory nerves in multiple animal models of chronic pain. Due to technical limitations, less research has studied spontaneous activity in sensory neurons in vivo, in their natural cellular environment. Using Pirt-GCaMP6 mice in which a genetically-encoded Ca2+ indicator GCaMP6 is specifically expressed in >95% of all DRG neurons under the control of the Pirt promoter, we found that spontaneously-firing `clusters' of neurons formed within the DRG after spared nerve injury. Mice with these ectopically active cluster neurons had increased spontaneous pain behavior. We also obtained preliminary evidence that cluster firing may be related to abnormal sympathetic sprouting in the sensory ganglia. Clinically, some forms of pain are relieved by sympathetic blockade, and this is reflected in some preclinical models of neuropathic pain in which sympathetic blockade reduces evoked pain behaviors and sensory neuron abnormalities. In this multi-PI proposal, we will examine mechanisms for cluster firing in order to identify potential novel therapeutic targets for neuropathic pain. We will focus on spontaneous pain, an understudied but clinically important aspect of neuropathic pain. Our central hypothesis is that cluster firing is triggered by abnormal sympathetic inputs to sensory neurons, and that it underpins spontaneous paroxysmal pain in neuropathic pain models. The proposal is a new collaboration between the laboratories of Xinzhong Dong at Johns Hopkins University and Jun-Ming Zhang at the University of Cincinnati. The Dong lab has developed the method for recording calcium signals from multiple DRG neurons in vivo in mice, and discovered the cluster- firing phenomenon. The Zhang lab brings expertise in sympathetic regulation of pain, in ex vivo DRG electrophysiology, and in local application of drugs to the DRG or nerve injury site in rats in vivo. We will investigate our hypothesis with three specific aims: Aim 1. Characterize spontaneously firing cluster neurons that develop after peripheral nerve injury and determine their relationship to spontaneous pain behavior. Aim 2. Determine if sympathetic activation triggers cluster firing of the DRG neurons after peripheral nerve injury. Aim 3. Determine the mechanism through which sympathetic innervation affects cluster firing of DRG neurons in nerve-injured rodents. The proposed research is significant because it will examine mechanisms of spontaneous pain in neuropathic pain models, which are currently understudied, and expand our understanding of how sympathetic blockade relieves some types of pain, and why this may occur in only a subset of patients. By focusing on the peripheral nervous system, we will develop therapeutic targets likely to lack abuse potential.